Processes for preparing conjugated linoleic acid from conjugated linoleic acid esters

ABSTRACT

Processes for preparing conjugated linoleic acid are described wherein a conjugated linoleic acid lower alkyl ester is subjected to hydrolysis in the presence of an enzyme to form a hydrolyzate comprising a conjugated linoleic acid and a lower alkanol, wherein at least a portion of the lower alkanol is continuously removed; the hydrolyzate is separated into an organic phase and an aqueous/alcoholic phase; and the conjugated linoleic acid is separated from the organic phase.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to fatty acids and, more particularly,to a new process for the production of conjugated linoleic acid byenzymatic hydrolysis of its esters.

2. Prior Art

Linoleic acids with conjugated double bonds, which are commerciallyavailable as “CLA” (conjugated linoleic acids), are physiologicallyactive and are used as food additives. Conjugated linoleic acid isnormally produced from triglycerides which have a high percentagecontent of—normally unconjugated—linoleic acid, such as thistle orsunflower oil for example. The triglycerides are isomerized in thepresence of basic catalysts or enzymes and then saponified. Adisadvantage in this regard is that, on the one hand, the saponificationstep yields many unwanted waste materials and, on the other hand, largequantities of alkalis are required, which can quickly result incorrosion in the reactors used. To avoid this, linoleic acid alkylesters have more recently been used as preferred starting materials and,in a first step, are isomerized to the CLA esters and then saponified.However, even this process is not entirely convincing because it is alsoattended by disadvantages, such as for example poor yields, drasticreaction conditions, unwanted secondary products and long reactiontimes.

Accordingly, the problem addressed by the present invention was toprovide a process for the production of conjugated linoleic acid whichwould reliably avoid the above-mentioned disadvantages of the prior art.

BRIEF SUMMARY OF THE INVENTION

The present invention relates, in general, to fatty acids and, moreparticularly, to new processes for the production of conjugated linoleicacid by enzymatic hydrolysis of conjugated linoleic acid esters.

The present invention relates to a process for the production ofconjugated linoleic acid, in which

-   (a) conjugated linoleic acid lower alkyl esters are hydrolyzed with    water in the presence of enzymes with continuous removal of alcohol,-   (b) the hydrolyzate is separated into an organic phase and an    aqueous/alcoholic phase and-   (c) the organic phase containing the conjugated linoleic acid is    freed from unreacted conjugated linoleic acid lower alkyl esters.

It has surprisingly been found that enzymatic hydrolysis with continuousremoval of alcohol leads to fatty acids that are free from unwantedsecondary products. High yields are obtained, the process involves mildreaction conditions and uses catalysts which meet all environmentalcompatibility requirements. In addition, if alcohol is removedcontinuously from the hydrolysis reactor itself during the hydrolysisprocess, a much faster conversion is achieved in a one-step process.

DETAILED DESCRIPTION OF THE INVENTION

Conjugated Linoleic Acid Lower Alkyl Esters

Starting materials for the process according to the invention arelinoleic acid lower alkyl esters which preferably correspond to formula(I):R¹CO—OR²   (I)where R¹CO is the acyl group of a linoleic acid containing conjugateddouble bonds and R² is a linear or branched alkyl group containing 1 to4 carbon atoms. In one particular embodiment, conjugated linoleic acidmethyl and/or ethyl esters are used.Enzymes

Typical—but not limiting—examples of suitable enzymes are lipases and/oresterases of microorganisms selected from the group consisting ofAlcaligenes sp., Aspergillus niger, Candida antarctica A, Candidaantarctica B, Candida cylindracea, Chromobacterium viscosum, Rhizomucormiehei, Penicilium camemberti, Penicilium roqueforti, Porcine pancreas,Pseudomonas cepacia, Pseudomonas fluorescens, Rhizopus javanicus,Rhizopus oryzae, Thermomyces lanugenosus and mixtures thereof. Lipasesand esterases from the organisms Alcaligenes, Candida, Chromobacterium,Rhizomucor, Pseudomonas, Rhizopus and Thermomyces are preferred becausethey are particularly active. The enzymes are generally used in the formof dilute suspensions or water-based concentrates. The lipases/esterasesmay also be immobilized on carrier material and re-used in so-calledrepeated batches.

Hydrolysis

The hydrolysis of the fatty acid alkyl esters is preferably carried outat mild temperatures in the range from 20 to 80° C., preferably in therange from 30 to 70° C. and more particularly in the range from 35 to60° C. with continuous removal of the lower alcohol, i.e. normallymethanol or ethanol, under reduced pressure, the preferred temperaturebeing determined by the activity optimum of the enzymes used.

A) A suitable hydrolysis process is a batch process in which a constantwater content—normally between 30 and 70% by weight—is maintained in thereactor by subsequent additions of water. The reaction is normallycarried out at a temperature of 30 to 50° C. and under a reducedpressure of 20 to 60±5 mbar. In this batch process, an alcohol/watermixture is continuously removed (“stripped”).

B) Another suitable hydrolysis process is a batch process in which wateris continuously introduced and an alcohol/water mixture is continuouslyremoved (“stripped”). The water content in the reactor in this processis usually low (0 to 20% by weight). The reaction is normally carriedout at a temperature of 50 to 70° C. and under a reduced pressure of 20to 60±5 mbar.

C) An alternative, but equally suitable, hydrolysis process is amultistage process without continuous removal of the alcohol component.On termination of the enzymatic hydrolysis, the water phase, which alsocontains large parts of the water-soluble short-chain alcohol, isseparated from the organic phase and a fresh water phase is added.Typically, the water phase is changed 1 to 3 times. The reaction isnormally carried out at a temperature of 20 to 70° C. and at a watercontent of 50 to 75%. The hydrolysis may be carried out with immobilizedenzyme, which may be re-used in each hydrolysis stage, and withnon-immobilized enzyme. In that case, fresh enzymes has to be added ineach hydrolysis stage.

Working Up

After the hydrolysis, the water/alcohol phase is separated from theorganic phase which is worked up, i.e. unreacted alkyl ester is removedfrom the valuable product. Different conversion rates are obtainedaccording to the duration of the hydrolysis. The reaction may beterminated early, for example at a conversion of only. 60% by weight, sothat fatty acids and fatty acid esters have to be subsequentlyseparated. However, it may also be terminated at a conversion of >90% byweight, preferably >95% by weight or even >99% by weight, so thatsubsequent separation is unnecessary. Separation may be carried out bydistillation or by saponification of the free fatty acid and subsequentphase separation. However, complete hydrolysis of the conjugatedlinoleic acid esters (conversion >99%) under mild reaction conditions isparticularly preferred in order to avoid changes in the isomercomposition.

EXAMPLES Example 1

Selection of Suitable Lipases.

15 Batches each containing 4 g conjugated linoleic acid ethyl ester and6 g water in a closable reaction vessel were simultaneously stirred atroom temperature on a multi-stirrer plate. Quantities of 40 mg ofcommercially available lipases or esterases were added to the batches.Samples are taken after reaction times of 2 h and 22 h. The organicphase containing fatty acid ethyl ester and enzymatically hydrolyzedfatty acid were separated and analyzed. The conversion was determinedvia the acid value. The results are set out in Table 1.

TABLE 1 Lipases and esterases used Acid value Conversion EnzymeMicroorganism Manufacturer 2 h 22 h 2 h 22 h Chirazym L-10 Alcaligenessp. Roche 21 41 11.5 20.5 Lipase A Aspergillus niger Amano 6 16 3 8Novozym 868 Candida antarctica A Novozymes 5 6 2.5 3 Novozym 525 Candidaantarctica B Novozymes 52 62 26 30 Lipomod 34 Candida cylindraceaBiocatalysts 45 61 22.5 30 Lipase LP Chromobacterium viscosum AsahiKasei 45 60 22.5 30 Novozym 388 Rhizomucor meihei Novozymes 8 11 4 5.5Lipase G Penicilium camemberti Amano 15 38 7.5 19 Lipase R Peniciliumroqueforti Amano 6 6 3 3 Lipase L115P Porcine pancreas Biocatalysts 6 63 3 Lipase PS Pseudomonas cepacia Amano 46 57 23 28.5 Lipase AKPseudomonas fluorescens Amano 26 53 13 26.5 Lipomod 36 P Rhizopusjavanicus Biocatalysts 21 38 11.5 19 Lipase F-AP 15 Rhizopus oryzaeAmano 12 18 6 9 Lipolase T1 100 Thermomyces lanugenosus Novozymes 38 5319 26.5

All the lipases and esterases tested were found to be active in thehydrolysis of the fatty acid esters. However, microorganisms of theAlcaligenes, Candida, Chromobacterium, Penicilium, Pseudomonas, Rhizopusand Thermomyces type are preferred. The reaction without removal ofethanol under the above conditions continued to an equilibrium of ca.30% by weight free fatty acid.

Example 2

Hydrolysis of Short-chain Conjugated Linoleic Acid Methyl Esters withContinuous Stripping of Water and Methanol

Hydrolysis Test using Process A)

400 g conjugated linoleic acid methyl ester, 200 g water and 20 gCandida antarctica B lipase immobilized on polypropylene were introducedinto a heatable flask. The reaction was carried out with a distillationbridge surmounting the flask under a reduced pressure of 60 mbar and ata temperature of 60° C. Water was continuously pumped into the flask ata flow rate of 0.5 ml/min. and a water content of 30 to 40% was adjustedin the flask. The conversion of the reaction was determined via the acidvalue. On termination of the reaction, the reaction mixture was filteredoff from the immobilized enzyme and the organic phase was separated fromthe aqueous phase. An acid value of 200 corresponded to a 100%conversion. The results are set out in Table 2.

TABLE 2 Conversion after different reaction times with stripping ofwater and methanol Reaction time [h] Acid value Conversion [%] 0 0 0 296.4 48.2 8 139.2 69.6 24 189.5 94.7 48 198.8 99.4

According to analysis of the acid value, a conjugated linoleic acid inthe form of a clear, pale yellowish colored liquid was obtained with aconversion of >99% after a reaction time of 48 hours.

The isomer pattern of the enzymatically hydrolyzed CLA was compared withthe starting substrate CLA methyl ester by gas chromatographic analysis.

TABLE 3 Comparison of the isomer pattern after enzymatic hydrolysisAnalysis CLA Me, crude CLA FFA, crude C16:0 3.8 4.3 C18:0 2.0 2.4 C18:116.8 17 C18:2 1.9 2 C18:2 c9, 11t 37.5 37 C18:2 t10, c12 36.8 36.7 C18:2cc isomers 0.8 1 C18:2 tt isomers 0.5 0.8 Acid value 198.8

Within the limits of measurement inaccuracy, the enzymatic hydrolysisdid not produce any significant change in the isomer pattern.

Example 3

Hydrolysis of Short-chain Conjugated Linoleic Acid Methyl Esters withContinuous Stripping of Water and Methanol

Hydrolysis Test using Process B)

100 g conjugated linoleic acid methyl ester, 10 g water and 5 g Candidaantarctica B lipase immobilized on polypropylene were introduced into aheatable flask. The reaction was carried out with a distillation bridgesurmounting the flask under a reduced pressure of 60 mbar and at atemperature of 60° C. Water was continuously pumped into the flask at aflow rate of 0.25 ml/min. (Example 3A) and 0.5 ml/min. (Example 3B).Added water was quickly distilled off so that the water content in thereactor was low (<20%) throughout the reaction. The conversion of thereaction was determined via the acid value. The reactions wereterminated after 24 h (partial conversion) and the immobilized enzymewas filtered off from the reaction mixture. The organic phase was thenseparated from the aqueous phase. An acid value of 200 corresponded to a100% conversion. The results are set out in Table 4.

TABLE 4 Conversion after different reaction times with stripping ofwater and methanol Reaction Acid value Conversion [%] Acid valueConversion [%] time [h] Example 3A Example 3A Example 3B Example 3B 0 00 0 0 2 51.5 25.7 62.3 31.1 4 71.2 35.6 84.4 42.4 6 87.0 43.5 100.2 50.18 100.0 50.0 112.2 56.1 24 153.0 76.5 167.1 83.5

Conversions of 76.5% and 83.5% were obtained after 24. h, depending onthe quantity of water added. The amount of distillate in Example 3A was315 g after 24 h and, in Example 3B, 584 g after 24 h.

Example 4

Hydrolysis of Short-chain Conjugated Linoleic Acid Ethyl Esters withContinuous Stripping of Water and Ethanol

Hydrolysis Test using Process A)

100 g conjugated linoleic acid ethyl ester, 100 g water and 10 gThermomyces lanugenosus lipase immobilized on polypropylene wereintroduced into a heatable flask. The reaction was carried out with adistillation bridge surmounting the flask under a reduced pressure of 30mbar and at an external temperature of 60° C. Water was continuouslypumped into the flask at a flow rate of 0.5 ml/min. and a water contentof 40 to 60% was adjusted in the flask. The temperature inside thereactor was kept at ca. 40° C. The conversion of the reaction wasdetermined via the acid value. On termination of the reaction, theimmobilized enzyme was filtered off from the reaction mixture and theorganic phase was separated from the aqueous phase. An acid value of 200corresponded to a 100% conversion. The results are set out in Table 5.

TABLE 5 Conversion after different reaction times with stripping ofwater and ethanol Reaction time [h] Acid value Conversion [%] 0 0 0 459.1 29.6 20 114 57 45 166 83

According to analysis of the acid value, a conjugated linoleic acid inthe form of a clear, colorless liquid was obtained with a conversion of83% after a reaction time of 45 hours.

Example 5

Hydrolysis of Short-chain Conjugated Linoleic Acid Methyl Esters byMultistage Hydrolysis

Hydrolysis Test using Process C)

3 Batches each containing 20 g conjugated linoleic acid methyl esterbased on the sunflower oil and 40 g water were weighed into closedflasks. Quantities of 1 g immobilized lipase were then added and themixtures were stirred for 5 h at room temperature on a magnetic stirrerplate. The enzyme immobilizates were then filtered off and the organicphase was separated from the aqueous phase. Another 40 g water was addedto the organic phase and the enzyme immobilizates filtered off werere-added to the reaction solution. After reaction overnight at roomtemperature, the enzyme immobilizates were again filtered off and theorganic phase was separated from the aqueous phase. 40 g water was addedto the organic phase and the enzyme immobilizates filtered off werere-added to the reaction solution. After another 5 h at roomtemperature, the reaction was terminated.

The following enzyme immobilizates were used:

-   5A) 1 g Novozym 435-   5B) 1 g Candida antarctica B lipase immobilized on macroporous    polypropylene

The conversion of the reaction in the individual stages was determinedvia the acid value. An acid value of 200 corresponded to a 100%conversion. The results are set out in Table 6.

TABLE 6 Conversion in multistage hydrolysis Acid Con- Acid Con- AcidReaction value version value version value Conversion time [h] Ex. 5AEx. 5A Ex. 5B Ex. 5B Ex. 5C Ex. 5C 0 0 0 0 0 0 0 Stage 1, 100.4 50.2%81.8 40.9% 74.5 37.3% after 5 h Stage 2, 142   71% 127 63.5% 117.2 58.6%after 16 h Stage 3, 165.5 82.8% 154.9 77.5% 152.4 76.2% after 5 h

Example 6

Hydrolysis of Short-chain Conjugated Linoleic Acid Ethyl Esters byMultistage Hydrolysis

Hydrolysis Test using Process C)

2 Batches each containing 20 g conjugated linoleic acid ethyl esterbased on thistle oil and 40 g water were weighed into closed flasks. 200mg non-immobilized lipase and 1 g immobilized lipase were then added.The batches were treated as described in Example 5. 200 mg freshnon-immobilized enzyme was added in each hydrolysis stage. The followingenzymes were used:

-   6A) 200 mg Lipomod 34 (Candida cylindracea lipase) per stage-   6B) 1 g Novozym 435 (Chromobacterium viscosum lipase) per stage

The conversion of the reaction in the individual stages was determinedvia the acid value. An acid value of 200 corresponded to a 100%conversion, The results are set out in Table 7.

TABLE 7 Conversion in mutistage hydrolysis Reaction time Acid valueConversion [%] Acid value Conversion [%] [h] Example 6A Example 6AExample 6B Example 6B 0 0 0 0 0 Stage 1, 56.6 28.3 101.9 51 after 5 hStage 2, 83.9 42 123 61.5 after 16 h Stage 3, 122 61 143 71.5 after 5 h

1. A process comprising: (a) subjecting a conjugated linoleic acid loweralkyl ester to hydrolysis in the presence of an enzyme to form ahydrolyzate comprising a conjugated linoleic acid and a lower alkanol,wherein at least a portion of the lower alkanol is continuously removed;(b) separating the hydrolyzate into an organic phase and anaqueous/alcoholic phase; and (c) separating the conjugated linoleic acidfrom the organic phase.
 2. The process according to claim 1, wherein theconjugated linoleic acid corresponds to the general formula (I):R¹CO—OR²  (I) wherein R¹CO represents a linoleic acid acyl group havingconjugated double bonds and R² represents an alkyl group having from 1to 4 carbons.
 3. The process according to claim 1, wherein the enzymecomprises a compound selected from the group consisting of esterases,lipases and mixtures thereof.
 4. The process according to claim 1,wherein the enzyme comprises at least one microorganism selected fromthe group consisting of Alcaligenes., Aspergillus niger, Candidaantarctica A, Candida antarctica B, Candida cylindracea, Chromobacteriumviscosum, Rhizomucor miehei, Penicilium camemberti, Peniciliumroqueforti, Porcine pancreas, Pseudomonas cepacia, Pseudomonasfluorescens, Rhizopus javanicus, Rhizopus oryzae, and Thermoinyceslanugenosus.
 5. The process according to claim 2, wherein the enzymecomprises at least one microorganism selected from the group consistingof Alcaligenes., Aspergillus niger, Candida antarctica A, Candidaantarctica B, Candida cylindracea, Chromobacterium viscosum, Rhizomucormiehei, Penicilium camemberti, Penicilium roqueforti, Porcine pancreas,Pseudomonas cepacia, Pseudomonas flucrescens, Rhizopus javanicus,Rhizopus oryzae, and Thermomyces lanugenosus.
 6. The process accordingto claim 1, wherein the enzyme comprises at least one microorganismselected from the group consisting of Candida antarctica B,Chromobacterium viscosum, and Thermomyces lanugenosus.
 7. The processaccording to claim 2, wherein the enzyme comprises at least onemicroorganism selected from the group consisting of Candida antarcticaB, Chromobacterium viscosum, and Thermomyces lanugenosus.
 8. The processaccording to claim 1, wherein the hydrolysis is carried out at atemperature of from 20 to 80° C.
 9. The process according to claim 2,wherein the hydrolysis is carried out at a temperature of from 20 to 80°C.
 10. The process according to claim 6, wherein the hydrolysis iscarried out at a temperature of from 20 to 80° C.
 11. The processaccording to claim 7, wherein the hydrolysis is carried out at atemperature of from 20 to 80° C.
 12. The process according to claim 1,wherein the hydrolysis is carried out to a conversion of 60% by weight.13. The process according to claim 1, wherein a constant water contentof from 30 to 70% by weight is maintained during the hydrolysis and atleast a portion of the water/lower alkanol phase is continuously removedby application of a vacuum of from 20 to 60±5 mbar.
 14. The processaccording to claim 1, wherein water content is adjusted to from 0 to 20%by weight during the hydrolysis and at least a portion of thewater/lower alkanol phase is continuously removed by application of avacuum of from 20 to 60±5 mbar.
 15. The process according to claim 1,wherein the hydrolysis is carried out in two or more stages and a watercontent of from 50 to 75% by weight is used in each stage.
 16. Theprocess according to claim 1, wherein the hydrolysis is carried out to aconversion of greater than 90% by weight.
 17. The process according toclaim 1, wherein the hydrolysis is carried out to a conversion ofgreater than 99% by weight.